Transcriptional regulation occurs as a result of many different signaling pathways that lead to an activation of transcriptional factors that regulate promoter transcriptional activity. Alterations in gene expression leading to transcription and transcriptional regulation can be induced by a wide variety of inducers, stress, insults, environmental changes, and during development and other biological processes such as cellular growth, innate immunity, and metabolism. Reporter gene technology is a widely used and important approach to assess promoter activity or expression and changes in gene expression as a result of transcriptional regulation. The term “reporter” refers to a gene product that can be easily measured when it is fused to transcriptional control elements, such as those in promoters, and that “reports” the effect of a signalling cascade or experimental conditions on gene expression in living cells. The promoter is best known to be located immediately upstream of a transcription start site of a gene and often comprises a core promoter, is generally within 50 bp of the transcription site where the pre-initiation complex forms along with a general transcription machinery which assembles including polymerase II, and an extended promoter that can contain specific regulatory sequences elements (Cooper et al., 2006).
Regulated gene technologies including reporter gene technology is important both in academic basic scientific research and in pharmaceutical industry research. For example, it is used to monitor transcriptional activity of a specific promoter of interest or a cis-acting sequence of interest in health, disease, or therapeutic intervention research setting. The specific promoter of interest can be synthesized de novo or purposely altered, for example, by introducing specific mutations. Reporter gene technology is also used to assess the activity of transacting factors on specific promoters or sequences.
Regulated gene technologies including reporter gene technology is widely used in drug industry to discover and assess modulators, e.g., inhibitors, of the activity of specific promoters, specific cis-acing sequences, or of trans-acting factor activity. A drug discovery assay based on reporter gene technology is based on the measurement of transcription activity of a detectable reporter that is operably linked to promoters or specific cis-acting sequence elements that are activated by signaling cascades allowing a screening for compounds that modulate the transcriptional activity. The transcriptional reporter system can be studied for the effect of a new gene product or drug candidate on a particular signal transduction pathway.
Notable examples of cis-acting elements are, but not limited to, NFκB elements, AP-1 elements, IFN-stimulated response elements (ISRE), metal response elements, c-myc response elements, p53 response elements, calcium response elements, and many others. Intracellular receptors such as hormone receptors, e.g. glucocorticoid receptor, estrogen receptor, and androgen receptor, can also mediate transcription and can reach the nucleus either in its native state or modified by a specific signaling event.
Many of the regulatory transcriptional elements are used with reporter assays in a drug discovery program. For the purpose of providing an example, NFκB is one of these important elements in inflammation and cancer. This protein is a member of the rel family of transcription factors that regulate several important physiological processes, including immune responses, inflammation, cell growth, apoptosis, and tumorigenesis. As a result, the NF-κB signaling pathway has been increasingly seen as a promising target for pharmacological intervention, especially in models of inflammation or cancer, where the pathway is often constitutively active (Calzado et al., 2007). Many different stimuli have been identified which activate the NF-κB pathway such as the pro-inflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-1 (IL-1). There are many signaling pathways that regulate transcription that can be targeted by drugs. These signaling events result in modification and activation of transcriptional factors that subsequently act on the transcriptional machinery. Thus, the transcription reporter assays are important in pharmaceutical industry.
However, when post-transcriptional assessment is desired instead of assessing transcriptional effects, different regulated gene technologies including reporter gene technology have to be utilized, namely systems without or with minimal interference of transcriptional effects.
Thus, there is a need in the art for improved means and methods in the field of regulated gene technologies.
Thus, the object of the present invention is to provide means and methods that allow studying (post-)transcriptional regulation, in particular an improved assessment of post-transcriptional effects.